Protocols used in Molecular Biology

186 Pages·2020·5.221 MB·English

by  Sandeep Singh, Dhiraj Divakar

Protocols used in Molecular
Biology
Edited by
Sandeep Kumar Singh
Indian Scientific Education and Technology Foundation,
Lucknow-226002, India
Centre of Biomedical Research, SGPGI Campus,
Lucknow-226014, India
&
Dhiraj Kumar
Rameshwar College, B.R.A. Bihar University,
Muzaffarpur-842001, India
Protocols Used in Molecular Biology
Editor: Sandeep Kumar Singh
ISBN (Online): 9789811439315
ISBN (Print): 9789811439292
© 2020, Bentham eBooks imprint.
Published by Bentham Science Publishers Pte. Ltd. Singapore. All Rights Reserved.
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CONTENTS
FOREWORD ........................................................................................................................................... i
PREFACE ................................................................................................................................................ iii
LIST OF CONTRIBUTORS .................................................................................................................. iv
CHAPTER 1 ISOLATION OF GENOMIC DNA FROM PLANT TISSUES ................................ 1
(cid:51)(cid:68)(cid:79)(cid:79)(cid:68)(cid:89)(cid:76)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)
INTRODUCTION .......................................................................................................................... 1
Principle .................................................................................................................................. 1
Sample Collection and Storage Conditions ............................................................................ 2
CTAB DNA Extraction Buffer: 100 ml .................................................................................. 3
T10E1 Buffer .......................................................................................................................... 4
DNA Isolation Protocol .......................................................................................................... 4
Purification .............................................................................................................................. 4
Useful Tips and Suggestions for Obtaining Optimum Results ............................................... 5
CONSENT FOR PUBLICATION ................................................................................................ 5
CONFLICT OF INTEREST ......................................................................................................... 5
ACKNOWLEDGEMENTS ........................................................................................................... 6
REFERENCES ............................................................................................................................... 6
CHAPTER 2 RNA ISOLATION PROTOCOL FROM CELLS AND TISSUES ........................... 7
(cid:51)(cid:68)(cid:79)(cid:79)(cid:68)(cid:89)(cid:76)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)
INTRODUCTION .......................................................................................................................... 7
Precautions to be Taken Before the Experiment ..................................................................... 8
I. Isolation of RNA Using TRIzol Method ............................................................................. 8
Principle .................................................................................................................................. 8
Materials Required .................................................................................................................. 9
Steps ........................................................................................................................................ 9
II. Isolation of RNA Using the CTAB Method ....................................................................... 9
Materials Required .................................................................................................................. 9
Steps ........................................................................................................................................ 10
III. Hot-Phenol RNA Extraction Method ............................................................................... 10
Materials Required .................................................................................................................. 10
Steps ........................................................................................................................................ 11
IV. QIAGEN RNeasy Kit Method .......................................................................................... 11
DNase Treatment .................................................................................................................... 12
Storage of RNA ....................................................................................................................... 12
Determining RNA Quality and Quantity ................................................................................ 12
Formaldehyde Agarose Gel Electrophoresis .......................................................................... 13
Materials Required .................................................................................................................. 13
Preparation of Formaldehyde Agarose Gel ............................................................................. 13
Preparation of RNA Samples for Electrophoresis ................................................................... 13
CONSENT FOR PUBLICATION ................................................................................................ 13
CONFLICT OF INTEREST ......................................................................................................... 14
ACKNOWLEDGEMENTS ........................................................................................................... 14
REFERENCES ............................................................................................................................... 14
CHAPTER 3 ANALYZING GENE EXPRESSION THROUGH REAL TIME PCR WHILE
NEO-TISSUE REGENERATION USING DEVE- LOPED TISSUE CONSTRUCTS .................... 15
(cid:39)(cid:76)(cid:89)(cid:68)(cid:78)(cid:68)(cid:85)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:55)(cid:68)(cid:85)(cid:88)(cid:81)(cid:3)(cid:48)(cid:76)(cid:81)(cid:82)(cid:70)(cid:75)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:87)(cid:92)(cid:68)(cid:89)(cid:85)(cid:68)(cid:87)(cid:3)(cid:55)(cid:85)(cid:76)(cid:83)(cid:68)(cid:87)(cid:75)(cid:76)(cid:15)(cid:3)(cid:53)(cid:88)(cid:83)(cid:76)(cid:78)(cid:68)(cid:3)(cid:54)(cid:76)(cid:81)(cid:75)(cid:68)(cid:15)(cid:3)(cid:54)(cid:75)(cid:88)(cid:69)(cid:75)(cid:68)(cid:81)(cid:78)(cid:68)(cid:85)
(cid:36)(cid:81)(cid:68)(cid:81)(cid:71)(cid:15)(cid:3)(cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:57)(cid:76)(cid:89)(cid:72)(cid:78)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:51)(cid:68)(cid:81)(cid:71)(cid:72)(cid:92)(cid:15)(cid:3)(cid:36)(cid:85)(cid:88)(cid:81)(cid:3)(cid:53)(cid:68)(cid:90)(cid:68)(cid:87)(cid:15)(cid:3)(cid:54)(cid:80)(cid:76)(cid:87)(cid:68)(cid:3)(cid:42)(cid:88)(cid:83)(cid:87)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:81)(cid:77)(cid:72)(cid:72)(cid:89)
(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:60)(cid:68)(cid:71)(cid:68)(cid:89)(cid:15)(cid:3)(cid:51)(cid:68)(cid:90)(cid:68)(cid:81)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:39)(cid:88)(cid:69)(cid:72)(cid:92)(cid:3)and(cid:3)(cid:51)(cid:85)(cid:68)(cid:71)(cid:72)(cid:72)(cid:83)(cid:3)(cid:54)(cid:85)(cid:76)(cid:89)(cid:68)(cid:86)(cid:87)(cid:68)(cid:89)(cid:68)
INTRODUCTION .......................................................................................................................... 16
BASICS OF RT-PCR ..................................................................................................................... 17
Merits of RT-PCR ................................................................................................................... 18
PCR Cycling ........................................................................................................................... 19
RT-PCR Master Mix Components .......................................................................................... 20
Thermostable DNA Polymerase ............................................................................................. 21
Deoxyribonucleotidetriphosphates (dNTPs) ........................................................................... 21
Divalent Cation Especially Magnesium Ions .......................................................................... 21
Template ................................................................................................................................. 21
RT-PCR Primer Design .......................................................................................................... 22
Primer Designing .................................................................................................................... 22
Basics of Primer Designing .................................................................................................... 23
RT-PCR Fluorescence Detection Chemistry .......................................................................... 23
SYBR Green-based Detection ................................................................................................ 24
Mechanism of Action .............................................................................................................. 24
Advantages .............................................................................................................................. 24
Limitations .............................................................................................................................. 24
TAQMAN probe Based Detection .......................................................................................... 25
Mechanism of Action .............................................................................................................. 25
Advantages .............................................................................................................................. 26
Limitations .............................................................................................................................. 27
RT-PCR Experimental Design and Gene Expression Profiling ............................................. 27
Setting up a Reaction Mixture ................................................................................................ 28
Basic PCR Protocol and Establishment of Reaction Cycle .................................................... 29
Application of RT-PCR .......................................................................................................... 30
CHALLENGES ............................................................................................................................... 31
CONSENT FOR PUBLICATION ................................................................................................ 32
CONFLICT OF INTEREST ......................................................................................................... 32
ACKNOWLEDGEMENTS ........................................................................................................... 32
REFERENCES ............................................................................................................................... 32
CHAPTER 4 A MODIFIED WESTERN BLOT PROTOCOL FOR ENHANCED SENSITIVITY
IN THE DETECTION OF A TISSUE PROTEIN ............................................................................... 35
(cid:54)(cid:68)(cid:70)(cid:75)(cid:70)(cid:75)(cid:76)(cid:71)(cid:68)(cid:3)(cid:49)(cid:68)(cid:81)(cid:71)(cid:3)(cid:53)(cid:68)(cid:76)(cid:15)(cid:3)(cid:48)(cid:68)(cid:79)(cid:79)(cid:76)(cid:78)(cid:68)(cid:85)(cid:77)(cid:88)(cid:81)(cid:68)(cid:3)(cid:53)(cid:68)(cid:82)(cid:3)(cid:42)(cid:72)(cid:71)(cid:71)(cid:68)(cid:15)(cid:3)(cid:58)(cid:68)(cid:79)(cid:76)(cid:68)(cid:3)(cid:61)(cid:68)(cid:75)(cid:85)(cid:68)(cid:15)(cid:3)(cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:88)(cid:80)(cid:76)(cid:87)(cid:85)(cid:68)(cid:3)(cid:54)(cid:72)(cid:81)
(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:51)(cid:68)(cid:92)(cid:68)(cid:79)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:49)(cid:72)(cid:72)(cid:85)(cid:68)(cid:77)(cid:3)(cid:55)(cid:76)(cid:90)(cid:68)(cid:85)(cid:76)(cid:15)(cid:3)(cid:53)(cid:68)(cid:78)(cid:72)(cid:86)(cid:75)(cid:3)(cid:46)(cid:17)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:92)(cid:68)(cid:3)(cid:51)(cid:85)(cid:68)(cid:87)(cid:68)(cid:83)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)
INTRODUCTION .......................................................................................................................... 35
SAMPLE PREPARATION ........................................................................................................... 36
GEL ELECTROPHORESIS ......................................................................................................... 37
BLOTTING ..................................................................................................................................... 38
WASHING, BLOCKING AND ANTIBODY INCUBATION ................................................... 39
QUANTIFICATION ...................................................................................................................... 40
TROUBLESHOOTING ................................................................................................................. 40
CONCLUSION ............................................................................................................................... 42
CONSENT FOR PUBLICATION ................................................................................................ 42
CONFLICT OF INTEREST ......................................................................................................... 42
ACKNOWLEDGEMENTS ........................................................................................................... 42
REFERENCES ............................................................................................................................... 42
CHAPTER 5 IMMUNOHISTOCHEMISTRY AS AN IMPORTANT TECHNIQUE IN
EXPERIMENTAL AND CLINICAL PRACTICES ............................................................................ 44
(cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:70)(cid:75)(cid:70)(cid:75)(cid:76)(cid:71)(cid:68)(cid:3)(cid:49)(cid:68)(cid:81)(cid:71)(cid:3)(cid:53)(cid:68)(cid:76)(cid:15)(cid:3)(cid:54)(cid:68)(cid:88)(cid:80)(cid:76)(cid:87)(cid:85)(cid:68)(cid:3)(cid:54)(cid:72)(cid:81)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:58)(cid:68)(cid:79)(cid:76)(cid:68)(cid:3)(cid:61)(cid:68)(cid:75)(cid:85)(cid:68)(cid:15)(cid:3)(cid:49)(cid:72)(cid:72)(cid:85)(cid:68)(cid:77)
(cid:55)(cid:76)(cid:90)(cid:68)(cid:85)(cid:76)(cid:15)(cid:3)(cid:36)(cid:76)(cid:77)(cid:68)(cid:93)(cid:3)(cid:36)(cid:17)(cid:3)(cid:49)(cid:68)(cid:76)(cid:78)(cid:15)(cid:3)(cid:36)(cid:81)(cid:68)(cid:80)(cid:76)(cid:78)(cid:68)(cid:3)(cid:48)(cid:76)(cid:86)(cid:85)(cid:68)(cid:15)(cid:3)(cid:54)(cid:75)(cid:76)(cid:78)(cid:75)(cid:68)(cid:3)(cid:37)(cid:75)(cid:68)(cid:85)(cid:68)(cid:87)(cid:76)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:92)(cid:68)(cid:3)(cid:51)(cid:85)(cid:68)(cid:87)(cid:68)(cid:83)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)
INTRODUCTION .......................................................................................................................... 44
DIRECT TECHNIQUE ................................................................................................................. 45
NEW DIRECT TECHNIQUE (ENHANCED POLYMER ONE STEPS TRAINING
METHOD) ....................................................................................................................................... 45
TWO-STEP INDIRECT TECHNIQUE ....................................................................................... 46
POLYMER CHAIN TWO STEP INDIRECT TECHNIQUE ................................................... 46
UNLABELED ANTIBODY ENZYME COMPLEX TECHNIQUES (PAP AND APAP) ...... 46
IMMUNOGOLD SILVER STAINING TECHNIQUE (IGSS) .................................................. 47
THE PRINCIPLE OF IHC ............................................................................................................ 48
TISSUE PREPARATION .............................................................................................................. 48
ANTIGEN RETRIEVAL ............................................................................................................... 50
A. Proteolytic Enzyme Digestion ........................................................................................... 50
B. Heat Mediated Antigen Retrieval Techniques ................................................................... 51
ANTIGEN-ANTIBODY INTERACTION ................................................................................... 51
AVIDIN–BIOTIN COMPLEX ...................................................................................................... 52
HAPTEN LABELLING TECHNIQUE ....................................................................................... 52
DETECTION METHODS ............................................................................................................. 52
QUANTIFICATION OF THE IMMUNOHISTOCHEMICAL STAINING ........................... 54
APPLICATION AND IMPORTANCE ........................................................................................ 54
TROUBLES AND LIMITATIONS .............................................................................................. 55
TROUBLESHOOTING OF THE EXPERIMENTAL PROBLEMS ..................................... 55
IMMUNOENZYMATIC LABELLING PROTOCOL FOR LOCALIZA- TION OF
ASTROCYTES USING ANTI-GFAP ANTIBODY .................................................................... 55
FOR CRYOSTAT SECTIONS DAY 1 PROCESSING ........................................................ 55
DAY 2 PROCESSING ........................................................................................................... 56
IMMUNOHISTOCHEMICAL PROTOCOL FOR LABELLING ASTRO- CYTES USING
ANTI-GFAP BY IMMUNE-FLUORESCENCE METHOD ...................................................... 56
FOR CRYOSTAT SECTIONS DAY 1 PROCESSING ........................................................ 56
DAY 2 PROCESSING ........................................................................................................... 57
CONSENT FOR PUBLICATION ................................................................................................ 57
CONFLICT OF INTEREST ......................................................................................................... 57
ACKNOWLEDGEMENTS ........................................................................................................... 57
REFERENCES ............................................................................................................................... 57
CHAPTER 6 PROTOCOLS FOR THE DETECTION AND PROTEOME ANALYSIS OF THE
YELLOW MOSAIC VIRUS INFECTED SOYABEAN LEAVES .................................................... 60
(cid:37)(cid:68)(cid:83)(cid:68)(cid:87)(cid:79)(cid:68)(cid:3)(cid:46)(cid:72)(cid:86)(cid:68)(cid:89)(cid:68)(cid:3)(cid:51)(cid:68)(cid:89)(cid:68)(cid:81)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:68)(cid:83)(cid:68)(cid:87)(cid:75)(cid:85)(cid:88)(cid:71)(cid:88)(cid:3)(cid:46)(cid:68)(cid:81)(cid:68)(cid:78)(cid:68)(cid:79)(cid:68)
INTRODUCTION .......................................................................................................................... 60
METHOD ........................................................................................................................................ 61
Protein Extraction and Virus Detection .................................................................................. 61
Iso-electric Focusing ............................................................................................................... 62
SDS-PAGE/ 2nd Dimension ................................................................................................... 62
Colloidal Coomassie Staining ................................................................................................. 63
Image Analysis ........................................................................................................................ 64
CONSENT FOR PUBLICATION ................................................................................................ 64
CONFLICT OF INTEREST ......................................................................................................... 64
ACKNOWLEDGEMENTS ........................................................................................................... 64
ABBREVIATIONS ......................................................................................................................... 64
REFERENCES ............................................................................................................................... 64
CHAPTER 7 2D-DIGE A POWERFUL TOOL FOR PROTEOME ANALYSIS .......................... 67
(cid:54)(cid:88)(cid:71)(cid:75)(cid:76)(cid:85)(cid:3)(cid:46)(cid:17)(cid:3)(cid:54)(cid:75)(cid:72)(cid:78)(cid:75)(cid:68)(cid:85)(cid:15)(cid:3)(cid:45)(cid:68)(cid:76)(cid:3)(cid:42)(cid:82)(cid:71)(cid:75)(cid:72)(cid:77)(cid:68)(cid:3)and(cid:3)(cid:39)(cid:76)(cid:81)(cid:72)(cid:86)(cid:75)(cid:3)(cid:53)(cid:68)(cid:77)(cid:3)(cid:48)(cid:82)(cid:71)(cid:76)
INTRODUCTION .......................................................................................................................... 68
Methodology ........................................................................................................................... 68
Steps of the Experiment .......................................................................................................... 70
ADVANTAGES OF DIGE ............................................................................................................. 71
Applications of DIGE ............................................................................................................. 72
CONCLUSION ............................................................................................................................... 72
CONSENT FOR PUBLICATION ................................................................................................ 72
CONFLICT OF INTEREST ......................................................................................................... 72
ACKNOWLEDGEMENTS ........................................................................................................... 72
REFERENCES ............................................................................................................................... 72
CHAPTER 8 MOLECULAR TECHNIQUES FOR GENOTYPING .............................................. 74
(cid:54)(cid:75)(cid:68)(cid:79)(cid:76)(cid:81)(cid:76)(cid:3)(cid:42)(cid:88)(cid:83)(cid:87)(cid:68)(cid:15)(cid:3)(cid:54)(cid:82)(cid:80)(cid:68)(cid:79)(cid:76)(cid:3)(cid:54)(cid:68)(cid:81)(cid:92)(cid:68)(cid:79)(cid:15)(cid:3)(cid:54)(cid:88)(cid:85)(cid:72)(cid:86)(cid:75)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:60)(cid:68)(cid:71)(cid:68)(cid:89)(cid:3)and(cid:3)(cid:48)(cid:68)(cid:71)(cid:68)(cid:81)(cid:3)(cid:47)(cid:68)(cid:79)(cid:3)(cid:37)(cid:85)(cid:68)(cid:75)(cid:80)(cid:68)(cid:3)(cid:37)(cid:75)(cid:68)(cid:87)(cid:87) 74
INTRODUCTION .......................................................................................................................... 74
1. Saliva Collection and Storage ............................................................................................. 75
2. Preparation for Cell Lysis ................................................................................................... 76
3. RNA Removal ..................................................................................................................... 76
4. Protein and Lipid Removal ................................................................................................. 76
5. Isolation and Purification of Genomic DNA ...................................................................... 76
6. Rehydration of g DNA ........................................................................................................ 77
Several Protocols Have Been Developed to Isolate DNA from Saliva ................................... 77
Sample Collection ................................................................................................................... 77
DNA Extraction ...................................................................................................................... 78
DNA Evaluation ...................................................................................................................... 78
Methods for DNA Extraction from Whole Blood .................................................................. 79
Two-step Lysis ........................................................................................................................ 79
One-step Lysis ........................................................................................................................ 79
Isolating Genomic DNA Using Magnetic Beads .................................................................... 79
GENOMIC DNA ISOLATION OF DNA FROM BLOOD ........................................................ 80
Reagents Required .................................................................................................................. 80
Procedure ................................................................................................................................ 80
VISUALIZATION OF DNA THROUGH AGAROSE GEL ELECTRO- PHORESIS .......... 81
Materials Required .................................................................................................................. 81
METHOD ............................................................................................................................... 81
Range of separation of linear DNA of different get concentrations ....................................... 81
Reagents .................................................................................................................................. 83
6x Gel-loading Buffer .................................................................................................... 83
DNA Staining Solution (10mg/ml) ......................................................................................... 83
EDTA (0.5M pH 8) ................................................................................................................. 83
TAE (50X) .............................................................................................................................. 83
TBE (5X) ................................................................................................................................ 83
TPE (10X) ............................................................................................................................... 83
POLYACRYLAMIDE GEL ELECTROPHORESIS ................................................................. 83
Materials ................................................................................................................................. 83
Buffers and Solutions .................................................................................................... 83
METHOD ........................................................................................................................................ 84
POLYMERASE CHAIN REACTION ......................................................................................... 86
Materials Required .................................................................................................................. 86
Method .................................................................................................................................... 86
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ................................... 87
Reagents Required .................................................................................................................. 87
Method .................................................................................................................................... 87
CONSENT FOR PUBLICATION ................................................................................................ 87
CONFLICT OF INTEREST ......................................................................................................... 87
ACKNOWLEDGEMENTS ........................................................................................................... 88
REFERENCES ............................................................................................................................... 88
CHAPTER 9 SODIUM BISULFITE CONVERSION OF HUMAN GENOME FOR DNA
METHYLATION STUDIES .................................................................................................................. 89
(cid:36)(cid:68)(cid:86)(cid:87)(cid:75)(cid:68)(cid:3)(cid:48)(cid:76)(cid:86)(cid:75)(cid:85)(cid:68)(cid:3)and(cid:3)(cid:52)(cid:68)(cid:71)(cid:68)(cid:85)(cid:3)(cid:51)(cid:68)(cid:86)(cid:75)(cid:68)
INTRODUCTION .......................................................................................................................... 90
Mapping Whole Genome DNA Methylation Using Sodium Bisulfite Conversion of DNA
Followed by Next-generation Sequencing .............................................................................. 92
Sodium Bisulfite Conversion of DNA ............................................................................ 92
Bisulfite Conversion Protocol ................................................................................................. 93
CONCLUDING REMARKS ......................................................................................................... 95
CONSENT FOR PUBLICATION ................................................................................................ 95
CONFLICT OF INTEREST ......................................................................................................... 96
ACKNOWLEDGEMENTS ........................................................................................................... 96
REFERENCES ............................................................................................................................... 96
CHAPTER 10 CHROMATIN IMMUNOPRECIPITATION (CHIP) ............................................. 97
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OVERVIEW OF CHIP ASSAY .................................................................................................... 98
GENERAL DESCRIPTION OF CHIP PROTOCOL ................................................................ 99
1. Cross-Linking ..................................................................................................................... 99
2. Cell Lysis ............................................................................................................................ 100
3. Shearing of DNA ................................................................................................................ 100
4. Immunoprecipitation ........................................................................................................... 100
5. Reversal of Cross-links and Characterization of DNA ....................................................... 101
6. Analysis of DNA ................................................................................................................. 101
Requirements .......................................................................................................................... 101
Troubleshooting ...................................................................................................................... 105
Limitation ................................................................................................................................ 105
Variation ................................................................................................................................. 106
APPLICATION .............................................................................................................................. 109
CONSENT FOR PUBLICATION ................................................................................................ 110
CONFLICT OF INTEREST ......................................................................................................... 110
ACKNOWLEDGEMENTS ........................................................................................................... 110
REFERENCES ............................................................................................................................... 110
CHAPTER 11 OSTEOSARCOMA CELL CULTURE AND MAINTENANCE TO DETECT
THE APOPTOTIC EFFECT OF SOME PROMISING COMPOUNDS BY POTENT MARKERS
VIZ. DNA FRAGMENTATION AND CASPASE-3 ACTIVATION ................................................. 114
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INTRODUCTION .......................................................................................................................... 114
Osteosarcoma Cells Culture and Maintenance ....................................................................... 115
DNA Fragmentation ................................................................................................................ 115
Detection of Apoptosis via Caspase-3 Activity in Cancer Cells ............................................ 117